The broad long-term objective of this proposal is to understand the mechanisms of ion channel gating, a key molecular event that is known to regulate a variety of physiological and pathological processes. Studying the BK-type, voltage, Ca2+ and Mg2+ dependent K+ channel as a model system, the focus of this proposal is to investigate the molecular process in which voltage sensor movements, Ca2+ or Mg2+ binding are coupled to the opening of the activation gate through intramolecular interactions. The specific aims are: I. To elucidate the role of a cytosolic domain, the AC region, in Ca2+ dependent gating. II. To investigate the interactions between the bound Mg2+ and the voltage sensor. III. To examine effects of mutations in S6 on the function of the activation gate and the sensitivity to voltage, Ca2+ and Mg2+. The role of BK channels in human health is based on their activation by voltage, Ca2+ and Mg2+, and BK channels are being pursued as a therapeutic target for various diseases. A BK channel mutation that affects voltage and Ca2+ dependent activation is linked to epilepsy and paroxysmal dyskinesia. This application seeks to dissect the molecular mechanism of BK channel gating by these stimuli, which will provide insights into BK channel related diseases and a solid basis for therapeutic developments. Several studies in recent years have provided us with X-ray crystallographic structures of potassium channels Kv1.2, KvAP, and MthK that can serve as models for BK channels. Based on these structural models and other preliminary results, a multi-disciplinary approach, including electrophysiology, mutation, chemical modification, protein biochemistry and kinetic modeling will be used to achieve the specific aims. The BK-type potassium ion channel is important for brain function and blood circulation. This research investigates the mechanism of BK channel function. The results will improve our understanding of diseases caused by the malfunction of this channel such as epilepsy and hypertension, and facilitate the development of drugs treating various diseases such as neuronal ischemia, trauma and cognitive decline.